Melanoma occurs frequently with a significant contribution of environmental factors to its etiology. In addition to genetic mutations, aberrant epigenetic alterations also contribute to the malignant transformation of melanocytes. Recently, the loss of 5-hydroxymethylcytosine (5hmC) has been identified as a novel epigenetic mark for melanoma. The content of 5hmC is relatively high in healthy melanocytes but is gradually lost during the progression from benign nevi through malignant stages of primary and metastatic melanoma. 5hmC is converted from 5-methylcytosine (5mC) by TET (ten-eleven translocation) family dioxygenases. The global loss of 5hmC changes genome stability and genome-wide transcription patterns, leading to a cascade of phenotypic transformation from healthy melanocytes to malignant melanoma. Vitamin C was recently identified to induce the generation of 5hmC by acting as a cofactor for TET by our lab and confirmed by others. The uptake of vitamin C appears to be disrupted in melanoma cells. Therefore, it is plausible that vitamin C deficiency also contributes to the loss of 5hmC that is associated with the initiation and progression of melanoma. Preliminary results show that 0.1 mM vitamin C, a concentration in the range of healthy human plasma, increases the content of 5hmC in cultured melanoma cells (derived from radial growth phase (RGP), vertical growth phase (VGP) and metastatic phase) toward the level of healthy melanocytes. Based on these exciting findings, vitamin C treatment is hypothesized to help epigenetically reprogram melanoma cells toward healthy melanocytes by reestablishing normal 5hmC profiles in the genome. Two specific aims are proposed to test this hypothesis. Aim 1 is to examine whether vitamin C treatment reestablishes 5hmC profiles in the genome of melanoma cells toward healthy melanocytes. High-throughput sequencing will be used to characterize the vitamin C treatment-induced changes in 5hmC and transcription profiles of the whole genome in melanoma cells. Results of these experiments will determine whether vitamin C treatment causes 5hmC and transcriptome profiles in melanoma cells, particularly at RGP stage, toward healthy melanocytes. Aim 2 is to determine whether 5hmC reestablishment by vitamin C treatment decreases melanoma malignant phenotypes in vitro and tumorgenicity in vivo. In Vivo Imaging System (IVIS) and histological examinations will be conducted to analyze the formation of melanoma xenografts in Gulo knockout mice, which cannot synthesize vitamin C like humans, supplemented with or without vitamin C in the diet. Results of these experiments will determine whether vitamin C treatment reduces the malignancy of melanoma. Successful completion of this research will establish a potential role for vitamin C in reversing epigenomic alterations associated with melanoma, thereby prompting the development of novel epigenetic prevention and treatment for melanoma patients.